Spliceosome mechanism dissected at the single molecule level ABSTRACT: The spliceosome is a multi-megadalton RNA-protein complex that catalyzes in all eukaryotes the removal of introns and the ligation of exons during splicing of pre mRNAs. In humans, 94% of all pre-mRNAs undergo alternative splicing, which allows for the dynamic expression of various protein isoforms from a single gene through cell- and tissue-specific networks of regulated splicing events. It is estimated that up to 50% of all mutations leading to human disease act through disrupting the splicing code. Due to the availability of unique genetic and biochemical manipulation tools, the budding yeast Saccharomyces cerevisiae has long provided a central model system for dissecting the mechanism of eukaryotic pre-mRNA splicing. Despite 25 years of study, however, there is still little known about the compositional and conformational rearrangements, timing, and coordination associated with yeast spliceosome function. To address this challenge, we recently have developed single molecule fluorescence resonance energy transfer (smFRET) assays that have begun to dissect pre-mRNA conformational changes during splicing. In particular, we have identified a small, efficiently spliced yeast pre-mRNA, in which donor and acceptor fluorophores could be placed in the exons adjacent to the 5' and 3' splice sites, and have used it to show that the spliceosome operates close to thermal equilibrium. Here, we propose to follow up on this advance and begin to dissect the mechanism of splicing at the single molecule level. In Specific Aim 1, we will test the hypothesis that specific sets of conformational fluctuations lead to splicing, by adding to our tool set: (i) shuttered illumination combined with advanced hidden Markov modeling and in situ hybridization to faithfully track the conformational dynamics of single pre- mRNA substrate molecules over the entire time course of splicing; (ii) depletion-complementation approaches to introduce functionally active, fluorophore labeled small nuclear RNA (snRNA) and protein components of the spliceosome for coincidence analysis (CIA); (iii) covalent, small-tag fluorophore labeling approaches to non-invasively mark functional protein factors of the spliceosome; and (iv) an optimized affinity purification technique to isolate specific spliceosomal complexes with fluorophore labeled components for focused probing. In Specific Aim 2, we will follow up on our observation that our intron exhibits significant secondary structure, placing its flanking exons much closer than expected from their linear sequence distance. We will test the hypothesis that this secondary structure has a functional impact by introducing a systematic set of mutations that first impair, and then restore the predicted secondary structure, and by testing each mutant for splicing. In Specific Aim 3, we will dissect the mechanistic role of DExD/H-box helicase Prp2 in preparing the activated Bact. spliceosome for the first step of splicing by rearranging it into the B* complex with exposed pre- mRNA branch point. Taken together, these advances will pave the way for, over the funding period, extensive mechanistic studies of yeast splicing and for studying alternative splicing in humans in the longer term. PUBLIC HEALTH RELEVANCE: The spliceosome is the multi-megadalton RNA-protein complex present in all eukaryotes that catalyzes the removal of introns from pre-mRNAs. As a finely tuned process of great complexity and critical importance to the diversification of the proteome, it is thought that up to 50% of all mutations connected to human disease act through disrupting splicing. In this project, splicing in a model organism, baker's yeast, will be mechanistically dissected at an unprecedented single molecule level with the prospect of paving the way for studying human splicing diseases.